Pim-3慢病毒质粒构建及其表达鉴定

目的 构建Pim-3慢病毒质粒,鉴定其在卵巢癌SKOV3细胞中基因及蛋白水平的表达.方法 RT-PCR法提取Pim-3 cDNA,经T-A克隆及双酶切将其与慢病毒表达载体pCDH-EF1-MCS-T2A-copGFP重组;酶切鉴定及基因测序后,将阳性重组质粒导入病毒包装细胞293T中;收集病毒上清液转染SKOV3细胞,荧光显微镜下观察转染情况,应用RT-PCR及Western blot法分别检测Pim-3 mRNA及其蛋白.结果 RT-PCR法获得Pim-3基因,重组质粒酶切后片段大小及基因测序结果显示pCDH/Pim-3重组质粒构建成功.将感染重组质粒及空载体的293T细胞生产的病毒上清分别感染SKOV3细胞,感染重组质粒的SKOV3细胞中Pim-3 mRNA及蛋白表达水平均高于空载体细胞.结论 成功构建了pCDH/Pim-3重组慢病毒质粒,建立了Pim-3高表达SKOV3瞬转细胞模型,为探讨Pim-3在卵巢癌发生、发展中的作用及机制奠定实验基础.

Construction and identification of lentiviral vector expressing Pim-3 gene

Objective To construct the Pim-3 lentiviral vector recombinant plasmid and to detect the mRNA and protein expression levels in ovarian cancer SKOV3 cells.Methods Pim-3 cDNA was cloned by RT-PCR and inserted into pMD19-T （TA clone method）.XbaI and NotI were used to cut off the target gene and then inserted into the lentiviral vector pCDH-EF1-MCS-T2A-copGFP.The recombinant plasmid was transfected into 293T cells after being verified by double digestion and gene sequencing analysis.The culture supernatant of 293T cells was collected,filtered and added to the SKOV3 cells.Infection efficiency was observed by fluorescence microscopy,the mRNA and protein expression levels of Pim3 were detected by real-time PCR and Western blotting.Results Pim-3 cDNA was obtained by RT-PCR.Recombinant plasmid was successfully constructed after enzyme digestion and sequencing.The culture supernatant of 293T cells transfected with the recombinant plasmid and the empty vector were used to transfect the SKOV3 cells,the Pim-3 mRNA and protein expression levels in SKOV3 cells transfected with the recombinant plasmid were higher than those of the controls.Conclusion The pCDH/Pim-3 lentiviral vector recombinant plasmid is successfully constructed,and the Pim-3 is highly expressed in the SKOV3 cells,which will lay a foundation for exploration of the function and molecular mechanism of Pim3 in ovarian cancer.